1. Technical Field
The present invention relates to compositions and methods of treating neurodegeneration. More specifically, the present invention relates to compositions and methods of treating multiple sclerosis.
2. Background Art
Multiple sclerosis (MS) is a chronic disease in which the immune system of the individual attacks the central nervous system (CNS) and affects the nerve cells. When the immune system attacks the CNS, myelin and nerve fibers are damaged, making it difficult to communicate between the brain and the spinal cord. Nerve cells transmit electrical signals, called action potentials, through long fibers called axons, which are covered with an insulated, fatty substance, the myelin sheath. In an individual suffering from MS, the immune system attacks and damages the myelin sheath. This damage causes distorted or interrupted nerve signals traveling to and from the brain and spinal cord because the axons are no longer able to transmit signals effectively. The name “multiple sclerosis” is derived from multiple sclerosis scars (i.e. plaques or lesions) that are formed in the white matter of the spinal cord and brain.
There is no known specific cause of MS, and genetic, environmental, and viral infections can play a role in development of the disease. It is generally held that MS involves an immune-mediated process because an exact antigen has not yet been found. Correlations have been found in MS patients with environmental factors (such as being located in an area farther away from the equator causing lower levels of vitamin D production, toxins, diet) in a genetically susceptible individual. Viruses or other infectious agents can possibly also play a role in MS, such as Epstein-Barr virus responsible for mononucleosis, further discussed below. Combinations of these factors can also contribute to the disease. MS generally occurs for the first time between 15 and 50 years with a peak incidence in young adults, affecting twice as many women than men.
The disease can manifest with a wide range of neurological symptoms and can progress to total physical and cognitive disability, and is clinically assessed with EDSS (Expanded Disability Status Scale). There are many different symptoms of MS, including fatigue, numbness, walking and balance problems, bladder dysfunction, bowel dysfunction, vision problems, dizziness and vertigo, sexual dysfunction, pain, cognitive dysfunction, emotional changes, depression, and spasticity. MS can also take various forms, including relapsing-remitting (having clearly defined attacks of worsening neurological function) and progressive (having steadily worsening symptoms over time). The life expectancy for an individual with MS is approximately 5 to 10 years lower than that of a healthy person. Prognosis is difficult to predict and depends on many different factors. Generally, individuals experience a reduction in the regenerative capacity of their body with each new acute episode.
While there is no known cure for MS, there are several treatments available that are approved to reduce disease activity and disease progression for individuals who have relapsing MS. Some immunosuppressive or immunomodulatory treatments are given in quiescence periods between one episode and another. These include interferon beta-1a, interferon beta-1 b, glatiramer acetate, mitoxantrone, natalizumab, fingolimod, and teriflunomide. Non-steroidal anti-inflammatory drugs (NSAIDs) can also be given with each new acute episode (i.e. corticosteroids administered in the form of bolus injections, intramuscularly or orally). Several of these treatments are described below.
Interferon beta-1a (AVONEX®, Biogen, Idec) is made from a naturally occurring interferon. In controlled clinical trials in relapsing MS, those taking the medication had a reduced risk of disability progression, experienced fewer exacerbations, and showed a reduction in number and size of active lesions in the brain (as shown on MRI) when compared with the group taking a placebo. In a subsequent study of patients who had experienced a single demyelinating event in the optic nerve, spinal cord, or brainstem, and had lesions typical of MS on brain MRI, AVONEX® significantly delayed the time to a second exacerbation, and thus to a clinically definite diagnosis of MS. AVONEX® is administered by once-a-week intramuscular injection. Common side effects include flu-like symptoms. Related to interferon beta-1a is interferon beta-1 b (BETAFERON®, Bayer Health Care). Both of these drugs have anti-inflammatory properties and improve the integrity of the blood-brain-barrier.
Glatiramer acetate (COPAXONE®, Teva Pharmaceuticals) is an immunomodulator that is a random polymer of four amino acids found in myelin basic protein (glutamic acid, lysine, alanine, and tyrosine). Glatiramer acetate is thought to work as a decoy for the immune system. Glatiramer acetate has comparable efficacy to interferons. Administration is by subcutaneous injection. Side effects include flu-like symptoms and lumps at injection sites, and is not recommended during pregnancy. While quality of life can be improved, glatiramer acetate does not have an effect on the incidence of relapses.
Natalizumab (TYSABRI®, Biogen Idec) is another medication approved for MS treatment, as well as Crohn's disease. Natalizumab is a humanized monoclonal antibody against the cell adhesion molecule α4-integrin. Natalizumab is administered by intravenous infusion every 28 days. It is designed to impede movement of potentially damaging immune cells from the bloodstream across the blood-brain-barrier into the brain and spinal cord. Side effects include a risk of progressive multifocal leukoencephalopathy (PML) with fatal outcomes that can greatly outweigh the benefits of taking natalizumab.
Teriflunomide (AUBAGIO®, Genzyme) is an oral medication for relapsing-remitting MS. It is widely used in rheumatology in the form of its precursor levoflunamide. The main advantage of teriflunomide is that is a once-a-day oral capsule instead of an injection. However, there is potential hepatotoxicity with the need for monthly checks of liver transaminases and a very prolonged elimination time, up to 2 years. This treatment should not be used during pregnancy, and methods exist to accelerate the elimination of the drug in case of desired pregnancy.
Dimethyl fumarate (BG-12, TECFIDERA®, Biogen Idec) can be used in treatment of relapsing-remitting multiple sclerosis (RRMS), the most common form of MS. Dimethyl fumarate activates the Nrf2 pathway, reduces the activity and impact of inflammatory cells on the central nervous system (CNS), and can induce direct cytoprotective responses in CNS cells. These effects can enhance the CNS cells' ability to mitigate the toxic inflammatory and oxidative stress that play a role in MS pathophysiology. Phase III studies showed dimethyl fumarate reduced relapse rate and increased time to progression of disability. Administration is oral. Side effects include skin reactions (flushing/redness) and gastrointestinal disorders.
Alemtuzumab (LEMTRADA®, Genzyme) is a monoclonal antibody that binds to proteins (CD-52) on mature lymphocytes, targeting them for destruction. Alemtuzumab was used for years for treating chronic leukemia, but was withdrawn from the market to be reintroduced for multiple sclerosis with an increased price. It has high efficiency, but also frequent and serious side effects. Approximately 40% of patients develop thyroid autoimmune diseases, and hematologic and renal complications. The treatment therefore requires a precise monitoring program with monthly checks of the blood and urine examination, which must be planned for four years after the last infusion. Treatment includes a series of 5 daily infusions at the beginning and three infusions after a year. This therapy is not one that can be terminated or modified easily and it is not clear how to proceed in case of insufficient response to therapy. Alemtuzumab can be used only in patients with very active disease as defined in clinical or radiological exams, and not for those who have a stable path or no signs of active inflammation in MRI. Alemtuzumab is not currently approved in the US.
Laquinimod is an oral immunomodulator currently under investigation. It has been shown to slow the progression of disability and reduce rate of relapse.
Fingolimod is an oral immunomodulator that is restricted to patients with relapsing-remitting MS with high disease activity despite treatment with β-interferon or in patients with severe forms of the disease rapidly evolving. There are possible side effects for the liver, cardiac system, and eyes requiring special precautions.
In special situations, other immunosuppressive drugs (azathioprine, methotrexate, cyclophosphamide) can be used to block cell replication globally, thus slowing down the reaction of the immune system. Being potentially toxic drugs, also used in cancer chemotherapy, they are reserved for cases of MS with rapid progression and disabling that do not respond adequately to an immunomodulatory drug. The selection, prescription, and monitoring of these therapies requires special expertise.
Other treatments have been focused more on the effects of the Epstein-Barr virus in MS patients, generally from mononucleosis. By was of background, infectious mononucleosis (also referred to as “kissing disease”, for its transmissibility through saliva) is a highly contagious infectious viral illness, caused by the Epstein-Barr virus (EBV). The virus targets B cells and the course is acute, usually 4-6 weeks in duration. In developed countries the disease typically affects younger adults, with a prevalence in adolescents, while in developing countries the illness is more frequently observed in childhood, often in the first five years of life (Straus, et al 1993). Mononucleosis is so-named due to increased levels of mononuclear cells (lymphocytes and monocytes) and mononuclear cell properties.
Over 90% of the adult population has been infected by the virus and have developed antibodies against viral antigens. Again, peak infection rates are noted during early childhood in developing countries, while infection rates tend to be highest between 15 and 25 years of age in industrialized countries. The course may be asymptomatic or indistinguishable from that of a flu syndrome or pharyngitis with minor symptoms. Classic symptoms of mononucleosis tend to occur in affected younger patients, while affected adults typically experience a milder illness, characterized by fever, malaise and weakness, all characteristic symptoms of multiple sclerosis and rheumatoid arthritis. Several studies have demonstrated that in patients afflicted with autoimmune diseases such as systemic sclerosis, ulcerative colitis, systemic lupus erythematosus and to a lesser extent, those with rheumatoid arthritis, Sjögren's syndrome, ankylosing spondylitis and Crohn's disease, the presence of Epstein-Barr stimulates the formation of an autoantibody that may contribute to the maintenance of the inflammatory state (Draborg, et al 2013). Similar, but rare forms of autoimmune disease are caused by other infections such as Herpesvirus (CMV, HHV-6), other viral forms (adenovirus, HIV, HAV, Rubella), Streptococcus pyogenes and Toxoplasma gondii. It should be noted that several studies have provided evidence that mononucleosis is a risk factor for multiple sclerosis (Thacker, et al 2006).
In some individuals, likely predisposed by congenital or acquired factors that alter the immune response against the virus, the primary infection is unrestrained, and the Epstein-Barr virus continues to replicate causing chronic active infection (CAEBV) or very severe chronic active infection (SCAEBV). This syndrome has been variously described, because of its similarity to other ill-defined illnesses such as chronic fatigue syndrome and hemophagocytosis syndrome, both correlated with EBV (Eligio, et al 2010). In the majority of cases, acute illness lasts no more than four weeks without any complications; however, immunodeficiency mononucleosis can be very serious disease and lethal for immunocompromised patients. In any case, the symptoms can persist for months after healing.
Additionally, contrary to commonly held beliefs, patients with CAEBV are more frequently diagnosed with lymphoid malignancies originated by T cells, NK cells and B lymphocytes, as observed in post-transplant lymphoproliferative disease who are also found to be EBV positive. EBV in particular, and other infectious agents that can cause mononucleosis type responses (CMV, toxoplasmosis, hepatitis viruses, HIV), are among the recognized causes of chronic fatigue syndrome, which may also compromise the endocrine system (Glser et al 2012). The symptoms of infectious mononucleosis lasts about a month and is followed by a period of convalescence characterized by weakness, of varying duration and is not to be confused with chronic fatigue syndrome. The assumption is that intense weakness during the acute phase of mononucleosis places the patient at greater risk of developing this complication. Other rare complications include orchitis, myocarditis, pericarditis, genital ulcers, neutropenia and interstitial pneumonia. A similar pathogenesis of fulminant hepatitis is not considered, which is very rare and primarily observed in people with severe immune deficiencies.
Pathogenesis is as follows. After binding to its receptor, EBV penetrates the B lymphocyte. Its DNA exists within the cell in two forms: an episomial form in which the viral DNA remains detached from human genomic material, and an integrated form in which DNA is incorporated into the host genome, without a specific site of integration. After infection of B lymphocytes, two processes can occur: In the first response which is characteristic of most viral infections, a viral replicative cycle ensues, resulting in lysis of the infected cell. This process is followed by the release of new viral particles, which will continue to infect other cells. The second response, typical of EBV and of a few other viruses, suggests a state of latency during which the virus does not multiply within the cell. This latent period can last for a very long time and may explain why an individual exposed to EBV can accommodate a number of infected cells throughout most of their lifetime. Following infection by B cells, the viral genome governs the synthesis of certain proteins, called Epstein Barr Nuclear Antigens (EBNA). It was originally believed that EBNA consisted of only one protein. It is now known that EBNA consist of six different proteins, numbered from EBNA-1 to EBNA-6. These proteins interfere with the cell's DNA by changing the expression of several genes and permanently activating B cells that go through a process of indefinite proliferation (cellular immortalization). This phenomenon has been studied in vitro by reproducing human lymphocytes infected with the virus. The cells were found to proliferate indefinitely under the influence of the viral proteins EBNA, three membrane proteins (LMP1-2A-2B) and two types of non-polyadenylated RNA (EBER1 and EBER2). The cell line produced is called a lymphoblastoid cell line (LCL). Furthermore, two processes are likely to occur following the infection of b cells: The first process involves the beginning of a viral replicative cycle and the death of the infected cells by lysis. This is followed by the release of new viral particles that will go on to infect other cells. The second, typical of EBV and of a few other viruses, produces a state of latency during which time, the virus does not multiply within the cell. This latency can last for several years, and thus explains why an individual who has been exposed to the EBV can accommodate a number of infected cells indefinitely.
Based on the expression of viral proteins, and expression of cell surface markers, three programs of viral latency were identified:
Latency I is characterized by the expression of EBNA-1, Q by the promoter (Qp), the EBER 1.2 and LMP2A. In vivo, the EBV persists for life in memory B cells of a healthy carrier. In pathological conditions, the expression of these three genes characterizes the pathology of Burkitt's lymphoma and its corresponding cell lines.
Latency II is characterized by the expression of EBNA-1, Q by the promoter (Qp) and also of LMP1, LMP2A, EBV Ebers. There can also be an expression of LMP2B. Latency II has been observed in Hodgkin's disease, nasopharyngeal carcinoma, lymphoma nasal NK/T, and primary effusion lymphomas.
Latency III is characterized by transcription of all nine latent proteins. EBNA 1-2-3-4-5-6 are transcribed by the promoter Wp/Cp. Use of this promoter is the defining characteristic of latency III. Such cells are lymphoblastoid cell lines and some lines of Burkitt's lymphoma in prolonged culture. Latency III is also found in lymphomas associated with EBV infection in immunocompromised individuals.
It is therefore plausible to assume that a person whose immune system is compromised from immortalization by T and B lymphocytes present with the Epstein Barr Virus. The state of immortalization described above acts as a potential trigger for chronic, degenerative inflammation. Movement of b cells from the circulatory system to the lymph system, and consequently through the blood-brain barrier, would appear to trigger a chronic inflammatory process resulting in a demand for the production of reactive T lymphocytes; a likely consequence of the degeneration of B cells affected and changed as described above. The lymphatic system consists of a fluid that has been enriched with substances saturated by interstitial fluid due to the normal drainage of tissues. Infection or tissue damage may increase the production of fluid, rich in a number of substances (in particular antigens) that can trigger immune responses. Sites that are conducive to microbe entry such as the gastrointestinal tract are rich in dendritic cells and can capture antigens. These microbial antigens as well as several chemical inflammation mediators are then released into the bloodstream and make their way to the lymph nodes where the fluid is filtered and recirculated. This process is the activation of the adaptive immune response. It is thought that the ability to eradicate transcription errors at the endocellular level originates with the proliferation of T lymphocytes.
A team of researchers led by scientists at The Scripps Research Institute (TSRI), have discovered a family of proteins that connect the immune system to lipids in the human body. “This is the first time that shows someone how to blend the immune system and lipid metabolism”,—explains Luc Teyton, lead author of the study (2009). In the study, published in “Science”, Teyton and colleagues examined the T cells known as “natural killers” (NK) (2009). According to the authors, these cells play a key role in the immune system and are involved in autoimmune diseases such as diabetes and cancer, although the exact mechanism of is not yet understood. NK T cells represent a middle ground between innate and adaptive immunity: they are produced in the thymus and, once mature, stimulate an adaptive immune response. This is much like other T cell receptors, but without the normal antigenic variability. “Innate” receptors or NK cells have the ability to recognize certain lipids are on the outer surface of many bacteria such as Mycobacterium tuberculosis, the bacterium that causes TB. The NK T cells are activated when they bind to a surface protein called CD1 that produces an unidentified binder lipid. Once activated, these T cells secrete large amounts of proteins as interferon-gamma and interleukin-4, which in turn activate helper T cells to fight pathogens. The binding of CD1 with lipids is not limited to the immune response, but is used to maintain normal homeostasis of the body. Teyton, et al., realizing that an additional protein was necessary to transfer the lipid to the CD1 molecule, identified a family of genes that code for proteins, such as prosaposin, involved in several pediatric neurological diseases. Teyton, et al. found that eliminating the genes that encode for prosaposin disabled NK T cells from binding to CD1 molecules.
Although much is known about the molecular pathways involved in viral reactivation, what triggers reactivation in vivo is not precisely understood. The presumption is it that occurs when latently infected B cells respond to unrelated infections (because B cell receptor stimulation triggers reactivation in B-cell lines). A recent article has elucidated how an Epstein-Barr reactivation can have concomitant unrelated infections (Odumade et al 2011). Thus, basic protection against infection could, in principle, reduce the likelihood of relapse. The idea of utilizing antibiotics to produce a similar reaction is not a novel one. At the moment there are ongoing clinical trials aimed at evaluating the role of minocycline (an antibiotic) in treating multiple sclerosis.
Statins, widely prescribed as agents capable of lowering cholesterol levels, can be an alternative treatment in the future in multiple sclerosis, to be used individually or in related therapies, since they have demonstrated potent immunomodulatory effects (Neuhaus, et al., 2005). Several studies have revealed the ability of statins to prevent and to reverse the chronic and relapsing experimental autoimmune encephalomyelitis, an experimental animal model of multiple sclerosis (Luccarini, et al. 2008). Furthermore, in vitro studies with human immune cells have shown the immunomodulatory action of statins comparable to that of interferon beta 1-b (Neuhaus et al., 2005). In vitro studies have revealed the ability of statins to reduce the expression of cell adhesion molecules induced by activation on T cells (Koh, Nippon Rinsho 2003), specifically inhibit the expression of integrin LFA-1 on T cells, ligand of ICAM-1, cell adhesion molecule expressed on the luminal surface of brain endothelial cells, thereby preventing the entry of inflammatory cells, like T cells, within the brain parenchyma. A reduction of matrix metalloproteinase-9 (MMP-9) a proteolytic enzyme that helps to promote “openness” of the blood-brain barrier and, therefore, boost transendothelial migration of inflammatory cells was observed in vitro. Statins at least in vitro, reduce the expression of chemokine receptors on T cells that is B (Koh C S, Nippon Rinsho 2003). The implication is that statins can be effective immunomodulatory agents that deserve consideration as a treatment of multiple sclerosis (C S Koh, Nippon Rinsho 2003). The science is promising, as a clinical study conducted on patients diagnosed with multiple sclerosis, and treated with simvastatin, revealed a significant decrease in the number of new lesions, as demonstrated by MRI gadolinium (Neuhaus O et al., 2005).
Acycloguanosine was considered for its ability as an anti-retroviral, its low toxicity, as well as its proven ability as an antifungal and a known preventive agent against Epstein Barr Virus, as indicated in 1993 by Lonnqvist, et al. Also, Vitamin D3, which is synthesized by keratinocytes via the stimulation induced by UV rays, acts as a hormone, and is able to perform an effective immune-stimulating effect, especially in subjects with chronic deficits.
There appears to be a positive interaction between minocycline and atorvastatin, as suggested by research on experimental autoimmune encephalomyelitis (EAE) induced in mice, wherein Luccarini et al (2008), demonstrated that combined treatment with atorvastatin and minocycline suppresses severity of EAE. Although this research has shown that this combination has the ability to reduce the severity of injuries subsisting in subjects suffering from a form of induced MS, it has never been tested on human subjects. While minocycline and atorvastatin provides a positive interaction, patients would still be at risk of the side effect of fungal infections due to the use of antibiotics.
Each of the above treatments have a number of serious side effects and induce a constant weakening of the body, without providing any form of real recovery or rebalancing of the body. Therefore, there remains a need for a treatment for multiple sclerosis that can reduce symptoms at all stages of the disease as well as reduce and eliminate existing lesions while eliminating side effects.